The present invention relates to a stable pharmaceutical formulation for the slow release of coagulation promoting substances for the treatment of blood coagulation disorders.
Hemophilia A is one of the most frequently occurring inherited coagulation disorders. Patients with hemophilia A are prone to frequent hemorrhages as a result of one or more misfunctions of the coagulation system. One of the causes of hemophilia is a shortage of Factor VIII (FVIII) in the blood. This problem can be treated with Factor VIII concentrates. However, in about 15% of the patients the occurrence results of Factor VIII neutralizing antibodies, so-called inhibitors, whereby a therapy with Factor VIII concentrates is hardly possible.
Two basic approaches have been described in the literature to protect FVIII from inactivation by inhibitors.
WO/80/01456 to Hemker discloses a pharmaceutical composition suitable for oral administration comprising FVIII incorporated within liposomes of 0.5-1.0 microns formed from phospholipids. The phospholipids have a net charge, and the FVIII is incorporated between the layers of the liposome. It is claimed that FVIII levels in the plasma remained above about 5% of the normal value for a period of 50 hours.
U.S. Pat. No. 4,348,384 to Horikoshi states that a composition as described in Heinker was prepared, but did not give satisfactory results. Therefore, Horikoshi incorporates a protease inhibitor into the liposome together with FVIII, in order to protect it from proteolysis. 3% of the normal plasma levels of FVIII were obtained over a period of 6 hours.
U.S. Pat. No. 5,013,556 to Woodle discloses a liposome composition for use in delivering various drugs via the bloodstream. The liposome contains between 1-20 mole percent of an amphipathic lipid derivatized with a polyalkylether. Here also, the drug compound is entrapped within the liposome. These liposome compositions are available commercially under the name of Stealth(copyright) vesicles (SUV""s, small unilamellar vesicles comprised of phospholipid and polyethylene glycol (PEG) covalently bound to phospholipid).
A further problem with this approach is that liposomes having a large diameter have a short half-life. Therefore, the liposomes must be downsized under high pressure, which can affect protein activities as in coagulation factors V and VIII.
In a second approach, Barrowcliffe, T. W., et al. (1983) J. Lab. Clin. Med. 101:34-43 teaches that mixing FVIII with phospholipid extracted from human and/or animal brain imparts significant protection to the FVIII in vitro. In this approach, the phospholipid is bound to the FVIII rather than encapsulating it. Kemball-Cook, G. and Barrowcliffe, T. W. (1992) Thromb. Res. 67:57-71, teaches that a negatively-charged phospholipid surface is necessary for FVIII binding. Negatively charged phosphatidyl serine and phophatidic acid were found to be highly active in binding to FVIII, while phosphatidyl choline was inactive. However, negatively-charged phospholipids are toxic, and those derived from brain tissue may carry pathogenic agents.
EP 689,428 discloses a liposome composition comprising liposomes having an outer surface layer of hydrophilic polymer chains. A polypeptide or polysaccharide effector molecule is covalently attached to the distal ends of the polymer chains by activation of the lipid anchor prior to effector coupling.
It is an object of the present invention to provide a pharmaceutical composition comprising a protein or polypeptide for therapeutic treatment. In particular, it is an object of the present invention to provide a pharmaceutical composition comprising FVIII for the treatment of blood coagulation disorders.
It is a further object of the invention to provide FVIII in a form having an extended half-life in the bloodstream.
It is a still further object of the invention to provide a method for treating patients suffering from blood coagulation disorders, particularly hemophilia, and most particularly those having FVIII inhibitors.
In one aspect of the present invention there is provided a pharmaceutical composition for parenteral administration comprising a therapeutically effective amount of coagulation factor VIII (FVIII) and substantially neutral colloidal particles, the particles comprising 1-20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer, the polymer carrying substantially no net charge, wherein the FVIII is not encapsulated in the colloidal particles.
The present invention is based on the surprising and unexpected finding that neutral phospholipids derivatized with a bio-compatible hydrophilic polymer can be used to bind FVIII and protect it from inhibitors in the bloodstream. This provides a significant advantage over the prior art compositions, since the phospholipids used are synthetic and non-toxic, and can therefore be used in vivo for therapeutic treatment. Furthermore, the liposome does not encapsulate the FVIII so that smaller sized liposomes can be used which have a longer half-life in vivo, since they are not removed by the reticuloendothelial system (RES). As will be described below in greater detail, FVIII interacts non-covalently with the polymer chains on the external surface of the liposomes, and no chemical reaction is carried out to activate the polymer chains, unlike the composition disclosed in EP 689,428.
In the present specification, the terms xe2x80x9csubstantially neutralxe2x80x9d and xe2x80x9csubstantially no net chargexe2x80x9d mean neither positively nor negatively charged. However, a very low measured charge within experimental error of zero is included within the meaning of the above terms.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d is to be understood as referring to an amount of FVIII which results in a level of FVIII in the bloodstream having a desired therapeutic effect. Such an amount can be experimentally determined by administering compositions comprising different amounts of FVIII and measuring the level in the blood at various times after administration.
The amphipathic lipid used to prepare the colloidal particles is preferably a phospholipid, and may be obtained from either natural or synthetic sources. A most preferred phospholipid is phosphatidylcholine, most preferably egg-phosphatidylcholine.
The biocompatible hydrophilic polymer may include polymers from the polyalkylether, polylactic or polyglycolic acid families. Preferably, the polymer is polyethylene glycol (PEG). The purpose of the polymer is to sterically stabilize the SUV""s, thus preventing fusion of the vesicles in vitro, and allowing the vesicles to escape adsorption by the RES in vivo. The polymer will preferably have a molecular weight of between about 1000 to about 5000 daltons, most preferably approximately 2000 daltons.
The colloidal particles will preferably have a mean particle diameter of between about 0.05 to about 0.4 microns, most preferably about 0.1 microns. This is to increase their circulation time in vivo and prevent their adsorption by the RES. The amphipathic lipid comprises approximately 1 to about 20 mole % of the particles, preferably approximately 1-5%, most preferably 5%.
A variety of known coupling reactions may be used for preparing vesicle forming lipids derivatized with hydrophilic polymers. For example, a polymer (such as PEG) may be derivatized to a lipid such as phosphatidylethanolamine (PE) through a cyanuric chloride group. Alternatively, a capped PEG may be activated with a carbonyl diimidazole coupling reagent, to form an activated imidazole compound. Other reactions are well known and are listed, e.g. in the aforementioned U.S. Pat. No. 5,013,556, whose contents are incorporated herein by reference.
The FVIII used in the composition of the invention is commercially available. It may be from a natural human source, or, preferably, it may be recombinantly prepared. Recombinant FVIII is commercially available, for example, Antihemophilic Factor (Recombinant), rFVIII-SQ (Pharmacia), and Kogenate, Miles Inc., Pharmaceutical Division, Elkhart, Ind., U.S.A., among other suppliers.
The composition of the invention is administered parenterally, preferably iv. The prior art compositions were intended for oral use only, due to side effects caused during injection by the liposome composition. The composition of the invention, on the other hand, is not toxic by injection, apparently due to the lack of charge, among other causes. Amounts of up to 0.5 gm/Kg body weight of colloidal particles according to the invention have been injected without detectable toxic symptoms. The dose is expected to be in the approximate range of 25-75 i.u./Kg. body weight. The particle to FVIII ratio (w/unit FVIII) will preferably be between about 0.1 mg/unit and about 10 mg/unit, and most preferably, approximately 1 mg/unit. Although the free form of FVIII:C has a half-life of less than 2 hours (FVIII measured by clotting activity) in mice, FVIII administered in the composition of the invention is expected to be effective for at least 24 hours, which is the period of effective activity of the coagulation promoting compound. The composition of the invention is expected to be effective in xe2x80x9con demandxe2x80x9d and prophylactic treatment of hemophilia patients, and particularly those patients who have developed FVIII inhibitor antibodies.
The effectiveness of FVIII contained in the composition of the invention may be determined by a chromogenic assay which determines FVIII activity by two consecutive steps: (1) the FVIII-dependent conversion of Factor X to Factor Xa in a coagulation-factor reagent composed of purified components, and (2) the enzymatic cleavage of a chromogenic Factor Xa substrate to yield a chromophore which can be quantified spectrophotometrically. Under appropriate assay conditions, there exists a linear relationship between the rate of Factor Xa formation and the FVIII concentration. In addition, FVIII activity may be determined by a one-stage clotting assay. This assay determines FVIII activity by the conversion of prothrombin to thrombin, which subsequently cleaves fibrinogen to form a clot composed of fibrin. FVIII activity in hemophillic mice may also be determined by measuring the survival of the mice following a tail cut.
In a further aspect of the invention, there is provided a pharmaceutical composition for parenteral administration comprising a therapeutically effective amount of a protein or polypeptide and substantially neutral colloidal particles, said particles comprising approximately 1-20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer, said polymer carrying substantially no net charge, wherein said protein or polypeptide is selected from the group consisting of: (a) proteins or polypeptides capable of externally binding said colloidal particles; and (b) proteins of polypeptides capable of binding polyethylene glycol (PEG), and wherein said protein or polypeptide is not encapsulated in said colloidal particles.
The term xe2x80x9cproteins or polypeptides capable of externally binding said colloidal particlesxe2x80x9d includes proteins and polypeptides which, similarly to FVIII, bind to membranes comprising phosphatidylcholine: phosphatidylserine (PC:PS) (see Haemostasis and Thrombosis. Arthur L. Bloom and Duncan P. Thomas (eds) (1987) Churchill Livingstone, pg. 179-180). Non-limiting examples of such proteins are coagulation factors such as prothrombin, Factor X and Factor V.
The term xe2x80x9cproteins or polypeptides capable of binding polyethylene glycolxe2x80x9d includes proteins and polypeptides which bind to PEG or derivatives of PEG by any non-covalent mechanism, such as ionic interactions, hydrophobic interactions, hydrogen bonds and Van der Waals attractions (Arakawa, T. and Timasheff, S. N. (1985) Biochemistry 24:6756-6762; Lee, J. C. and Lee, L. L. Y. (1981) J. Biol. Chem. 226:625-631).